Biofilm-forming antimicrobial-resistant pathogenic Escherichia coli: A one health challenge in Northeast India

This study aimed to investigate the prevalence of Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) in common food animals (cattle, goats, and pigs) reared by tribal communities and smallholder farmers in Northeast India. The isolates were characterized for the presence of virulence genes, extended-spectrum beta-lactamases (ESBL) production, antimicrobial resistance, and biofilm production, and the results were statistically interpreted. In pathotyping 141 E. coli isolates, 10 (7.09%, 95% CI: 3.45%–12.66%) were identified as STEC, 2 (1.42%, 95% CI: 0.17%–5.03%) as atypical-EPEC, and 1 (0.71%, 95% CI: 0.02%–3.89%) as typical-EPEC. None of the isolates were classified as ETEC. Additionally, using the phenotypic combination disc method (ceftazidime with and without clavulanic acid), six isolates (46.1%, 95% CI: 19.22%–74.87%) were determined to be ESBL producers. Among the STEC/EPEC strains, eleven (84.6%, 95% CI: 54.55%–98.08%) and one (7.7%, 95% CI: 0.19%–36.03%) strains were capable of producing strong or moderate biofilms, respectively. PFGE analysis revealed indistinguishable patterns for certain isolates, suggesting clonal relationships. These findings highlight the potential role of food animals reared by tribal communities and smallholder farmers as reservoirs of virulent biofilm-forming E. coli pathotypes, with implications for food contamination and zoonotic infections. Therefore, monitoring these pathogens in food animals is crucial for optimizing public health through one health strategy.


Introduction
Escherichia coli (E.coli) is a Gram-negative facultative anaerobic bacterium that is an innocuous occupant of the intestinal tract of warm-blooded animals and humans.However, E. coli has the potential to cause severe gastrointestinal (diarrhoeal) and extraintestinal diseases [1].E. coli strains incriminated in diarrhoeal cases are jointly referred to as diarrheagenic E. coli (DEC), a collection comprising enterohemorrhagic E. coli (EHEC), shigatoxigenic E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and enteroinvasive E. coli (EIEC).Among these strains, STEC/EHEC and EPEC constitute a significant zoonotic concern, which was established in multiple earlier studies [1,2].STEC colonises the large intestine via an attaching and effacing lesion.This lesion is caused by a bacterial type III secretion system that injects effector proteins into the intestinal epithelial cell, resulting in profound alterations to the host cell's architecture and metabolism and the close adherence of the bacteria.Shiga toxin (Stx), which exists in two main forms, Stx1 and Stx2, is responsible for severe diseases such as bloody diarrhoea and the hemolytic uremic syndrome.The stx genes are encoded on the chromosomes of temperate bacteriophages, and production and release of the toxin are highly dependent on phage induction [3].Ruminants, being the major animal reservoirs of STEC, are usually asymptomatic when colonized; however, a few STEC strains may cause diarrhoea in calves [4].
EPEC strains, classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC), are responsible for causing fatal diarrhoea characterized by the presence of attaching and effacing (A/E) lesions in the intestine.These lesions are mediated by intimin, which is encoded by the eae gene.Typical EPEC strains carry an EPEC adherence factor (EAF) plasmid containing the bfp operon, which encodes the bundle-forming pilus (BFP), while atypical EPEC strains lack the EAF plasmid [5].The central mechanism behind EPEC pathogenesis is the formation of A/E lesions, which involve the destruction of microvilli, intimate adherence of bacteria to the intestinal epithelium, pedestal formation, and aggregation of polarized actin and other cytoskeletal elements at sites of bacterial attachment.The genetic determinants responsible for the production of these A/E lesions are located on the locus of enterocyte effacement (LEE), which is a pathogenicity island containing genes encoding intimin, a type III secretion system, various secreted (Esp) proteins, and the translocated intimin receptor named Tir [5].Humans are the main natural reservoir of tEPEC, and they have been rarely isolated from animals.However, aEPEC strains have been repeatedly isolated from both diseased and healthy animals and humans [2].Several studies have demonstrated that STEC and EPEC strains from humans and animals share virulence characteristics and are clonally related [6].
The emergence of multi-drug resistance (MDR) in E. coli strains is complicating the treatment of many serious infections and is a serious public health concern.Earlier studies consistently reported an increase in antimicrobial resistance (AMR) among pathogenic E. coli strains isolated from both human and animal clinical samples [7].Particularly, the emergence of extended-spectrum beta-lactamases (ESBL) production and carbapenem resistance in E. coli strains is a serious threat to human health.The development of antimicrobial resistance in E. coli is usually due to the plasmid-mediated acquisition of antimicrobial resistant genes (ARGs).E. coli employs a variety of strategies to survive and persist in the environment, and several studies have documented the biofilm-forming capabilities of different E. coli pathotypes [8,9].The biofilm formation followed by the encasement of E. coli in a complex matrix can augment resistance to antibiotics and sanitising agents, leading to difficulty in eradicating and controlling them [9].Since MDR E. coli strains are becoming common and can form biofilms, it is essential that new, effective, and safe antimicrobial drugs be developed.These drugs should combat the difficulties of biofilm formation while being effective against resistant bacteria, such as those that produce ESBL and display carbapenem resistance.The prevalence of diarrhoea, especially in below-five-year-old children is 10.6% and 2.5% in Meghalaya and Assam (Northeastern states of India), respectively [10].But the prevalence of STEC, EPEC, and ETEC in food animals and their virulence potential are not well studied, and there is a paucity of studies.In this study, we examined rectal swabs obtained from commonly raised food animals (cattle, goat, and pig) in Northeastern India's tribal communities and smallholder farms located in Meghalaya and Assam.Our primary objective was to assess the prevalence of E. coli pathotypes (STEC, EPEC, and ETEC) and investigate potential correlations among virulence genes, phenotypic antimicrobial resistance, ARGs, biofilm-forming ability, and PFGE fingerprinting within these specific E. coli pathotypes.

Sample collection
During 2019-2020, a total of 150 rectal swabs were collected from apparently healthy cattle (n = 50), goats (n = 50), and pigs (n = 50) reared in backyard conditions in villages in Meghalaya (Ri Bhoi District) and Assam (Nagaon and Morigaon Districts) of Northeastern India (Fig. 1).All of these animals were raised in a semi-intensive housing system following conventional management practices.It is important to note that the animals received minimal or no antibiotic treatment, and specifically, antibiotics in the form of growth promoters were not administered to them.The studied households only had 1-2 animals per household, and feeding was usually managed with kitchen waste, locally available fodder, raw cereals, and vegetables.As the majority of pigs are raised in traditional small-holder systems, convenient sampling was conducted by obtaining one sample from each randomly chosen household or farm.The ethical approval for conducting the study with non-invasive sampling was obtained from the Institutional Animal Ethics Committee (IAEC) of ICAR RC NEH, Meghalaya, which is regulated by the Committee for Control and Supervision of Experiments on Animals (CCSEA) guidelines.The rectal swabs were aseptically collected with the help of a sample collector fixed with a cotton swab and sterile transport medium (HiMedia, Mumbai, India).The swabs, after soaking in the transport medium, were gently inserted into the rectum of the animals to collect the samples.The collected samples were transported to the laboratory under chilled conditions and immediately processed for the isolation of E. coli.

Isolation and identification of E. coli and PCR-based confirmation
The isolation and identification of E. coli were done as previously described (Milton et al., 2019).The collected samples were inoculated into MacConkey broth (HiMedia, India) and incubated at 37 • C for 24 h.After enrichment, the inoculum was streaked onto eosin methylene blue (EMB) agar (HiMedia, India) and incubated at 37 • C for 24 h.The colonies with a green metallic sheen were further streaked into nutrient agar slants (HiMedia, India) for further biochemical characterization (indole production, methyl red test, Voges-Proskauer test, and citrate utilisation test) and PCR confirmation.The isolates were grown in Luria Bertani broth (HiMedia, India), and genomic DNA was extracted employing the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).PCR analysis using coamplification of five genes, i.e., lacY [11], lacZ [12], cyd [11], phoA [13], and uidA [12], was done for specific confirmation of E. coli and differentiation from Shigella spp.and other species of Enterobacteriaceae.E. coli ATCC 25922 was used as the reference strain.

Pathotyping and detection of other virulence genes
For pathotyping the recovered E. coli isolates into STEC, EPEC, and ETEC, PCR screening of stx1 [14], stx2 [14], eae [15], bfp [15], stII [15], and lt [15] genes were done using previously described methods.Further, the presence of serotype-specific virulence genes such as wzx O104 , fliC H4 , rfb O157 , and fliC H7 [16] was also determined using PCR-based analysis.The presence of the hlyA gene was also detected using a previously described PCR protocol [17].The reference strains and field isolates from our previous studies were used as positive controls.

Analysis of biofilm production
Biofilm formation analysis was performed following a previously described microtiter plate-based method [26].The recovered pathogenic E. coli isolates were grown in LB broth (5 mL) at 35.5 • C overnight.The grown cultures (1.3 μL) were added to 130 μL of M9 broth with 0.8% glucose in polystyrene microtitre plates (96 wells/flat bottom) and incubated at 30 • C overnight, and the optical density (OD) of the wells was measured (λ = 620 nm).Then the broth was discarded, and wells were rinsed with sterile saline (150 μL) to eliminate nonadherent bacteria.After air drying for 20 min, 1% crystal violet solution (130 μL/well) was added and kept for 5 min.Later on, the colourant was removed, and the wells were washed three times with distilled water (180 μL) to remove excess stain.After airdrying for 1 h, 130 μL of absolute ethanol was added to solubilize the dye incorporated by the adhered bacterial cells.Finally, the OD of the test and control wells was read at λ = 540 nm.E. coli ATCC 25922 was used as a control in the experiment.The degree of biofilm formation was determined by applying a specific biofilm formation (SBF) index formula, SBF = (AB − CW) G, where AB is the OD 540 nm of stained adhered cells; CW is the OD 540 nm of stained control wells containing only M9 medium; and G is the OD 620 nm of growth in suspended culture.The isolates were classified as strong (≥1.10),moderate (0.70-1.09), weak (0.35-0.69), and negative (<0.35) biofilm producers, as described by Naves et al. [26].

PFGE fingerprinting and analysis
Pulsed-field gel electrophoresis (PFGE) was performed only on pathogenic E. coli strains (n = 13) as per the Centers for Disease Control and Prevention (CDC) guidelines using the PulseNet (24-26 h) standardized protocol.XbaI (50 U) restriction endonuclease (Thermo Scientific) was used to digest the DNA for each isolate, and CHEF DNA Size Marker-S.cerevisiae (BIO-RAD Labs, Hercules, CA) was used as a molecular size marker ranging from 225 kb to 2200 kb.The digested DNA was electrophoresed in the CHEF-DR® III System (BIO-RAD) with the following conditions: temperature of 14 • C; 120 • switch angle; 6 V/cm voltage; initial and final switch times of 6.76 s and 35.38 s, respectively; and a 20 h run time.The fingerprints were analyzed with Bionumerics v7.6 fingerprinting software (Applied Maths, Belgium).To check the genetic relatedness of the isolates based on the number of bands produced, cluster analysis employing the unweighted pair group method with averaging (UPGMA) was done to construct a dendrogram, unfolding the relationship among PFGE patterns (% similarity).The pathogenic E. coli strains were fitted to the same PFGE cluster if their index of similarity was ≥90%.

Statistical analysis
Statistical association between variables such as pathotypes, biofilm formation, and ESBL production was studied.The data was analyzed using Fisher's exact test to test hypotheses, with p-values calculated using MS-Excel.A heatmap was constructed using the "pheatmap" package with hierarchical clustering on R software (4.2.2 version).A contingency table of the attributes was prepared based on which a correlation plot with Spearman's rank correlation methods was generated using the "corrplot" package of R software (4.2.2 version).

Biofilm-producing ability
Using a microtiter plate-based method and a specific biofilm formation (SBF) index formula, we found that 12 out of 13 recovered pathogenic E. coli isolates were able to form biofilm, with an average absorbance (total) of 1.668 (Table 1).Among the biofilmproducing strains, eleven and one strain formed strong and moderate biofilms, respectively.The average absorbance of the strong biofilm-producing isolate was 1.854 (ranging from 1.109 to 2.064), and the absorbance of the moderate biofilm-producing isolate was 0.851.Except for one non-ESBL producer (G62), all five (LS3, G109, G12, G29, and B91) were observed as strong biofilm producers.

PFGE fingerprinting
The genetic relatedness of XbaI-digested E. coli isolates was determined by analyzing their PFGE patterns.Out of the 13 pathogenic E. coli isolates digested with XbaI, 9 different PFGE patterns (pulsotypes) were identified.However, one isolate (LS3, an STEC from a pig) could not be typed.Interestingly, STEC isolates (stx1 + and stx2 + ) from goats (G97 and G109) showed PFGE patterns with 100% similarity, indicating a close genetic relationship.In addition, three STEC isolates from goats and cattle, namely G29 (stx2 + , hlyA + ), G41 (stx2 + ), and B92 (stx2 + ), exhibited indistinguishable PFGE patterns, despite their differences in biofilm-producing ability (G97moderate biofilm and G109-strong biofilm) and antimicrobial resistance (B92-non-ESBL producer and G29-ESBL producer).The nine PFGE profiles were further classified into two groups (Group I and II) based on their 90% similarity, as depicted in the dendrogram shown in Fig. 2.This clustering implies that similar E. coli pathotypes tend to group together and share a clonal relationship.Overall, the findings suggest that similar pathotypes of E. coli (such as STEC) generate comparable PFGE patterns, regardless of their differences in biofilm formation or antimicrobial resistance.

Statistical analysis
Based on their phenotypic and genotypic traits, heatmap analysis showed that the isolates from the different hosts grouped together (Fig. 3).Furthermore, the correlation plot revealed significant positive associations among certain traits.Biofilm formation, cefpodoxime resistance, cefotaxime resistance, and the presence of the stx2 gene showed a strong positive correlation (Spearman's coefficient [ρ] > 0.92, p < 0.05) (Fig. 4).Similarly, as expected, ceftazidime resistance exhibited positive correlations with ESBL production and cefotaxime resistance (ρ > 0.7, p < 0.05).Additionally, the presence of the hlyA gene showed a positive correlation with ESBL production (ρ = 0.82, p < 0.05).

Discussion
This study investigated the presence of STEC, EPEC, and ETEC in the faeces of cattle, goats, and pigs reared by tribal people and smallholder farmers in Northeast India.Isolates were characterised for virulence genes, ESBL production, antibiotic resistance, and biofilm formation.The recovered isolates of pathogenic E. coli were fingerprinted with PFGE.On the basis of PCR-based pathotyping, 10 E. coli isolates were classified as STEC (7.09%), 2 as atypical EPEC (1.42%), and 1 as typical EPEC (0.71%); none of the isolates were typed as ETEC.In concordance with our study, a recent study from Northeast India has also documented a prevalence of 9.88% and 4.32% of STEC and EPEC, respectively, in small ruminants and associated samples.However, the same study documented a 12% prevalence of ETEC, whereas no ETEC was isolated in our study [27].Similar to the present study, a previous Indian study also described STEC followed by EPEC as the most prevalent pathotypes in neonatal calves [28].A previous study from Spain has also described the high prevalence of STEC and EPEC in goat's milk [29].In concordance with our study results, a lower rate of ETEC among food animals was previously described by many researchers around the world [30][31][32].The above observations are significant from a public health and zoonotic perspective, as the faecal carriage of E. coli pathotypes can contaminate foods of animal origin and can also transmit to humans through direct contact.We found that resistance to Amikacin was 7.7%, cefpodoxime was 77%, cefotaxime was 53.8%, and ceftazidime was 61.5%.Out of the isolates tested using the phenotypic combination disc method (ceftazidime with and without clavulanic acid), 46.1% (six isolates) showed positive results for ESBLs.The recovered pathogenic E. coli isolates did not harbour any of the tested antimicrobial resistance genes.It is quite evident from earlier observations that high antimicrobial resistance is usually associated with non-pathogenic or commensal strains [33,34].However, in some other studies, virulent microbes have displayed high AMR patterns as well [35,36].Further, in our study, the sampled animals were mostly reared by tribal communities and smallholder farmers in Northeastern India, who usually do not get much access to antibiotic treatment.
Twelve pathogenic E. coli isolates (92.3%) formed biofilm.The ability to form biofilms is crucial for any bacterial pathogen as it plays a significant role in infecting the host and contributing to its pathogenicity.For bacterial pathogens, biofilm formation facilitates long-term colonization and provides protection against antimicrobials and the host immune system [8].In multiple studies, the pathotypes of E. coli have been demonstrated to be strong and moderate biofilm formers [37,38].The results of a previous study have also indicated that the biofilm-forming ability could contribute to the persistence of STEC [37].
This study demonstrates that pathogenic E. coli strains of the same pathotype (STEC) exhibit indistinguishable PFGE patterns, regardless of their biofilm-forming capacity and antibiotic sensitivity.The PGFE analysis of these isolates establishes their identical nature.Previously, PFGE typing has been effectively used multiple times to decipher the genetic relatedness of E. coli pathotypes from various food animals [39,40].The strains shared high similarity based on PFGE, being clustered into two groups; however, nine PFGE patterns were observed.The hlyA positive isolates clustered together in spite of being from different sources.Similarly, isolates positive for either eae or bfp were placed in the same cluster.The isolates that harbored only the stx2 virulence gene comprised the biggest cluster.The remaining cluster was made up of isolates that harbored only the stx1 virulence gene and were all isolated from goats.Our findings support that the clustered pathotypes of E. coli are closely related; however, further investigations with larger numbers of  strains, including strains from clinically ill animals, may provide detailed insights for deriving more relevant epidemiological conclusions.
Heatmap analysis grouped isolates from different hosts by phenotypic and genotypic features.Biofilm development, cefpodoxime resistance, cefotaxime resistance, and stx2 gene presence were positively correlated (Spearman's coefficient >0.92; p < 0.05).Ceftazidime resistance was associated with ESBL generation and cefotaxime resistance.ESBL production and the hlyA gene were likewise positively correlated.However, a previous study from Iran reported a negative correlation the presence of the hlyA gene and ESBL production [41].In this study, no significant statistical association was observed between pathotypes and biofilm production.This is in agreement with a previous observation [42].
One limitation of the present attempt is that, due to limited resources, we were unable to investigate faecal samples from human contacts or animal owners to establish the zoonotic transmission potential of the isolated pathogens.Therefore, there is a need to undertake large-scale studies, including samples from animals, the environment, and tribal populations rearing animals, to elucidate the definite public health risk posed by the pathotypes of E. coli.Additionally, whole genome-based analysis of pathogenic E. coli strains from multiple sources is also required to study the real epidemiological link and impact.

Conclusions
The findings of the current study reveal that food animals raised by tribal communities and smallholder farmers in Northeastern India can serve as a reservoir of highly virulent biofilm-forming pathotypes of E. coli, specifically STEC/EPEC.A significant majority (92.3%) of the isolated STEC and EPEC strains were identified as biofilm formers, while 46.1% of the isolates exhibited ESBL production.Moreover, the application of PFGE fingerprinting demonstrated the circulation of identical clones among various livestock species and geographically distant sampling locations.STEC and EPEC strains are known to cause fatal and difficult-to-treat diseases in humans.Therefore, monitoring these pathogens in food animals is essential for optimizing public health through a one health strategy.Controlling STEC and EPEC in food animals ensures safer animal-derived food for consumers.

Fig. 1 .
Fig. 1.Map depicting the study area in Northeastern India, highlighting the districts where samples were collected from Assam and Meghalaya.

Table 1
Complete characterization profile of the pathogenic E. coli isolated from food animals.

Table 2
Antimicrobial resistance pattern of pathogenic E. coli isolated from food animals.